Interaction between Non-Heme Iron of Lipoxygenases and Cumene

Jan 28, 2004 - Cumene Hydroperoxide: Basis for Enzyme Activation, ... Cumene hydroperoxide was a reversible inhibitor of the reaction catalyzed by ...
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Interaction between Non-Heme Iron of Lipoxygenases and Cumene Hydroperoxide: Basis for Enzyme Activation, Inactivation, and Inhibition Ardeshir Vahedi-Faridi, Pierre-Alexandre Brault, Priya Shah, Yong-Wah Kim, William R. Dunham, and Max O. Funk, Jr.* Contribution from the Departments of Chemistry and Medicinal and Biological Chemistry, UniVersity of Toledo, 2801 West Bancroft Street, Toledo, Ohio 43606 Received October 16, 2003; E-mail: [email protected]

Abstract: Lipoxygenase catalysis depends in a critical fashion on the redox properties of a unique mononuclear non-heme iron cofactor. The isolated enzyme contains predominantly, if not exclusively, iron(II), but the catalytically active form of the enzyme has iron(III). The activating oxidation of the iron takes place in a reaction with the hydroperoxide product of the catalyzed reaction. In a second peroxide-dependent process, lipoxygenases are also inactivated. To examine the redox activation/inactivation dichotomy in lipoxygenase chemistry, the interaction between lipoxygenase-1 (and -3) and cumene hydroperoxide was investigated. Cumene hydroperoxide was a reversible inhibitor of the reaction catalyzed by lipoxygenase-1 under standard assay conditions at high substrate concentrations. Reconciliation of the data with the currently held kinetic mechanism requires simultaneous binding of substrate and peroxide. The enzyme also was both oxidized and largely inactivated in a reaction with the peroxide in the absence of substrate. The consequences of this reaction for the enzyme included the hydroxylation at Cβ of two amino acid side chains in the vicinity of the cofactor, Trp and Leu. The modifications were identified by mass spectrometry and X-ray crystallography. The peroxide-induced oxidation of iron was also accompanied by a subtle rearrangement in the coordination sphere of the non-heme iron atom. Since the enzyme retains catalytic activity, albeit diminished, after treatment with cumene hydroperoxide, the structure of the iron site may reflect the catalytically relevant form of the cofactor.

Introduction

Lipoxygenases constitute an important facet of polyunsaturated fatty acid metabolism in both plants and animals. The products of the reactions catalyzed by these enzymes are intermediates in the biosynthesis of numerous compounds in physiological signaling pathways. For example, in plants lipoxygenase inaugurates the wound-induced biosynthesis of the plant growth hormone jasmonic acid.1 Arachidonic acid metabolism in animals is governed by cyclooxygenases and lipoxygenases. The eicosanoid products of the lipoxygenase pathway include the leukotrienes, a family of paracrine hormones.2 Blocking the lipoxygenase pathway for therapeutic benefit in diseases such as asthma, atherosclerosis, and cancer is an active area of investigation.3 The lipoxygenases catalyze the regio- and steroselective peroxygenation of the methylene-interrupted unsaturated systems found in natural substrates such as linoleic, linolenic, and arachidonic acids. Remarkably, the products of the catalyzed reaction are conjugated hydroperoxides, compounds with cytotoxic properties.4 Therefore, the regulatory properties of the enzyme have important physiological consequences. The reaction catalyzed by lipoxygenases depends in a critical fashion (1) Feussner, I.; Wasternack, C. Annu. ReV. Plant. Biol. 2002, 53, 275-297. (2) Funk, C. D. Science 2001, 294, 1871-1875. (3) Julemont, F.; Dogne, J. M.; Laeckmann, D.; Pirotte, B.; DeLeval, X. Expert Opin. Ther. Pat. 2003, 13, 1-13. 2006

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on the presence of a unique non-heme iron cofactor.5 The active form of the enzyme contains iron(III). In the currently held mechanistic hypothesis, the enzyme abstracts hydrogen from the substrate in a step that has the properties of a hydrogentunneling reaction.6 The electron from a substrate C-H bond reduces the iron, while the proton is taken up by an iron-bound hydroxide. An intermediate free radical combines with molecular oxygen to produce a conjugated peroxyl radical which reoxidizes the iron and becomes protonated in the formation of product. Redox cycling of the iron atom during catalysis is firmly established.7 However, the enzyme as it is isolated, for example, from soybeans, contains predominantly if not exclusively iron(II).8 The only known oxidizing agent for lipoxygenase iron is the hydroperoxide product of its reaction, making kinetic time courses appear highly autocatalytic in nature.9 Under certain circumstances, lipoxygenases are sensitive to inactivation by fatty acid hydroperoxides, a self-inactivation reaction.10 Lipoxygenases are also inactivated by acetylenic (4) Schneider, C.; Tallman, K. A.; Porter, N. A.; Brash, A. R. J. Biol. Chem. 2001, 276, 20831-20838. (5) Solomon, E. I.; Brunold, T. C.; Davis, M. I.; Kemsley, J. N.; Lee, S. K.; Lehnert, N.; Neese, F.; Skulan, A. J.; Yang, Y. S.; Zhou, J. Chem. ReV. 2000, 100, 235-349. (6) Knapp, M. J.; Rickert, K.; Klinman, J. P. J. Am. Chem. Soc. 2002, 124, 3865-3874. (7) Funk, M. O.; Carroll, R. T.; Thompson, J. F.; Sands, R. H.; Dunham, W. R. J. Am. Chem. Soc. 1990, 112, 5375-5376. (8) Dunham, W. R.; Carroll, R. T.; Thompson, J. F.; Sands, R. H.; Funk, M. O. Eur. J. Biochem. 1990, 190, 611-617. 10.1021/ja0390855 CCC: $27.50 © 2004 American Chemical Society

Enzyme Activation, Inactivation, and Inhibition

analogues of their substrates.11 In this instance, inactivation was accompanied by conversion of a specific methionine residue in the enzyme to methionine sulfoxide. The hypothesis was that the acetylenic analogues were oxygenated to a reactive intermediate allenic hydroperoxide that could subsequently oxidize methionine, resulting in inactivation. Support for the link between sulfoxide formation and inactivation, however, has not been found. For example, the turnover-dependent inactivation of leukocyte 12-lipoxygenase was not significantly altered in any of three engineered proteins where the sulfides in candidate methionines were replaced by the less reactive aliphatic amino acid side chains of leucine or valine in site-directed mutagenesis experiments.12 Understanding the activation/inactivation chemistry of lipoxygenases is important since this could provide a means for controlling catalysis, both in the laboratory and for therapeutic purposes. For example, compounds that inhibit lipoxygenase by interfering with the oxidation/activation reaction have recently been discovered.13 To provide a basic understanding of the chemistry underlying the activation and inactivation of lipoxygenases by peroxides, we present the results of a study of the interaction of soybean lipoxygenases with cumene hydroperoxide. Lipoxygenase-1 was oxidized and largely inactiVated by this compound. We offer EPR spectroscopic, mass spectrometric, and crystallographic evidence for the chemistry that accounts for these effects. Because the enzyme retains significant catalytic activity following treatment with cumene hydroperoxide and the EPR spectroscopic features of the treated enzyme are identical to the product activated enzyme, the iron site in the cumene hydroperoxide treated enzyme may correspond to the active iron(III) form of the cofactor. Cumene hydroperoxide was also an inhibitor of the steady-state phase of the catalyzed reaction, but remarkably only at very high substrate concentration. Results and Discussion

Cumene Hydroperoxide Is an Inhibitor of the SteadyState Phase of Lipoxygenase Catalysis. When cumene hydroperoxide was included in substrate solutions at various concentrations under standard assay conditions (enzyme added last), a distinctive pattern of inhibition of the steady-state phase of the reaction was observed. At low substrate concentration, there was a small effect of the presence of cumene hydroperoxide on the rate of the catalyzed reaction. At higher concentrations of substrate, however, there was a major reduction in the rate even at relatively low concentrations of the peroxide (1 mM) of lipoxygenase-1 treated with an equimolar amount of 13-HPOD have proportionately less EPR visible iron than less concentrated solutions (250 µM).32 This “spontaneous reduction” of the iron(III) in lipoxygenase-1 was also observed during prolonged storage of solutions.23 The reversion of the iron(III) enzyme to the native form under the high-concentration conditions required for crystallization could (31) Knapp, M. J.; Seebeck, F. P.; Klinman, J. P. J. Am. Chem. Soc. 2001, 123, 2931-2932. (32) Petersson, L.; Slappendel, S.; Feiters, M. C.; Vliegenthart, J. F. G. Biochim. Biophys. Acta 1987, 913, 228-237. J. AM. CHEM. SOC.

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Figure 10. Fo - Fc electron density map showing spherical electron density centered at 2.05 Å for the iron atom. The electron density is contoured at 3σ. Table 2. Bond Distances and Angles for the Non-Heme Iron Sites in the Three-Dimensional Structures of Soybean Lipoxygenases Determined by X-ray Crystallography lipoxygenase-3, CmOOH 98 K

Fe-His-518 (NE2) Fe-His-523 (NE2) Fe-His-709 (NE2) Fe-Asn-713 (OD1) Fe-Ile-857 (OXT) Fe-Ile-857 (O) Fe-O22/OH

2.37 2.38 2.41 3.33 2.79 2.39 2.05

His-518 (NE2)-Fe-His-523 (NE2) His-518 (NE2)-Fe-His-709 (NE2) His-518 (NE2)-Fe-Asn-713 (OD1) His-518 (NE2)-Fe-Ile-857 (OXT) His-518 (NE2)-Fe-Ile-857 (O) His-518 (NE2)-Fe-O22/OH His-523 (NE2)-Fe-His-709 (NE2) His-523 (NE2)-Fe-Asn-713 (OD1) His-523 (NE2)-Fe-Ile-857 (OXT) His-523 (NE2)-Fe-Ile-857 (O) His-523 (NE2)-Fe-O22/OH His-709 (NE2)-Fe-Asn-713 (OD1) His-709NE2-Fe-Ile-857 (OXT) His-709 (NE2)-Fe-Ile-857 (O) His-709 (NE2)-Fe-O22/OH Asn-713 (OD1)-Fe-O22/OH Ile-857 (OXT)-Fe-Asn-713 (OD1) Ile-857 (OXT)-Fe-O22/OH Ile-857 (O)-Fe-Asn-713 (OD1) Ile-857 (O)-Fe-O22/OH a

93.8 108.2 68.8 158.1 135.8 85.7 109.7 159.9 108.1 91.4 118.4 86.2 63.4 111.2 128.9 52.5 90.0 85.4 94.4 53.9

lipoxygenase-1 100 K26

bond distances (Å) 2.21/2.24 2.34 2.29 2.87 2.28 3.51 2.11 bond angles (deg) 94.0 99.7 74.5 166.6 133.7 89.0 100.6 164.1 97.0 94.5 99.5 92.3 85.6 123.1 157.4 69.8 93.2 81.8 93.2 44.7

lipoxygenase-3 RT34

2.23 2.21 2.26 3.01 2.13 3.01 4.02 91.6 100.7 67.8 171.6 127.9 76.8 86.5 158.6 94.5 110.2 69.4 91.5 85.5 126.4 155.6 109.3 106.6 99.9 88.0 68.6

lipoxygenase-3, 13-HPOD RT29

2.23 2.24 2.28 2.28 2.05 3.07 2.01 84.5 98.9 77.9 178.2 133.7 90.1 89.2 161.5 94.3 107.5 82.7 87.9 82.4 125.1 167.3 102.9 103.4 88.4 89.0 49.5

Lipoxygenase-1 equivalent residues: His-499, His-504, His-690, Asn-694, and Ile-839.

account for the fact that crystals of the oxidized enzyme have been difficult to prepare. When preexisting crystals of lipoxygenase-3 were treated with cumene hydroperoxide and analyzed crystallographically, in addition to the oxidative modifications already described, a subtle rearrangement was observed in the coordination sphere of the iron atom relative to the native state. The new geometry is illustrated in Figure 10, and the bond distances and angles for the iron site are collected in Table 2. Values of angles and distances for other crystallographically determined lipoxygenase structures were included in the table for comparison purposes. The iron in the treated crystals was five-coordinate including three nitrogen ligands contributed by histidines and one oxygen ligand from the C-terminal isoleucine, usual features for lipoxygenase iron. Despite certain specific differences, the geometry closely resembles the one in the low-temperature structure of native lipoxygenase-1. For example, a superposition analysis for the iron and its six nearest neighboring atoms (His2012 J. AM. CHEM. SOC.

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518 NE2, His-523 NE2, His-709 NE2, Ile-857 O/OXT, Asn713 OD1, and OH) resulted in an rms deviation of 0.39 Å. The orientation of the coordinating C-terminal carboxylate group of Ile-857 was, however, significantly different in the iron environment for the protein in the cumene hydroperoxide treated crystals. Whereas the caboxylates in the native enzyme and the 13-HPOD product complex employ the oxygen atom designated OXT for iron coordination, the cumene hydroperoxide treated enzyme uses the oxygen atom designated O. While the two oxygen atoms may be chemically equivalent, they have distinguishable orientations in the three-dimensional structures. Also, the position of Asn-713 was beyond a bonding distance as was found for the structures of the native enzymes. In the 13-HPOD complex with lipoxygenase-3, Asn-713 becomes an iron ligand. The fifth ligand to iron in the cumene hydroperoxide treated crystals was a solvent molecule or hydroxide ion. There was spherical electron density in the 2Fo - Fc and the Fo -Fc omit map (contour levels 3σ) centered at 2.05 Å from the iron

Enzyme Activation, Inactivation, and Inhibition

(Figure 10). This value is consistent with the bond distance expected for iron(III) hydroxide. The 19 appropriate compounds in the Cambridge Crystallographic Data Base have an average iron(III) hydroxide bond distance of 1.96 ( 0.10 Å. The best analogy in a non-heme protein is iron(III) superoxide dismutase. The average iron hydroxide bond distance in the five crystallographically determined structures is 2.16 ( 0.18 Å.33 The current hypothesis for the mechanism of lipoxygenase catalysis invokes hydrogen abstraction from the substrate in a reaction that has the properties of hydrogen tunneling.6 In the process, the electron reduces the iron and the proton is thought to combine with iron-bound hydroxide. There is spectroscopic evidence for the existence of the iron hydroxide,34 and water molecules were found in the vicinity of the iron in the threedimensional structures of the native, iron(II) lipoxygenases: at 4.0 Å in lipoxygenase-3 at room temperature and at 2.01 Å in lipoxygenase-1 at 100 K.25,35 The structure reported here confirms the existence of iron hydroxide in the peroxide oxidized form of the enzyme. The five-coordinate iron site in the cumene hydroperoxide treated lipoxygenase-3 crystals is also remarkably similar to the iron environment found in E. coli galactose-1-phosphate uridyltransferase, which was described as a distorted square pyramidal geometry.36 A superposition analysis of the three histidine nitrogen atoms and two oxygen atoms in the coordination spheres for the two enzymes resulted in an rms deviation of 0.44 Å. In contrast to the situation in lipoxygenase-3, the sharp bond angle in the coordination sphere of iron in galactose-1phosphate uridyltransferase is achieved through bidentate ligation of the side chain of a glutamate residue. Remarkably, the iron in galactose-1-phosphate uridyltransferase was not involved in catalysis.37 Compounds with an iron configuration similar to the one found in the cumene hydroperoxide treated lipoxygenase-3 crystals have been produced previously in model studies for the mononuclear non-heme iron sites in various metalloproteins including lipoxygenase. For example, monomeric fivecoordinate complexes were obtained for iron(II) with a tris(pyrazolyl)borate and various acetate and benzoate ligands.38 The geometry was described as either square pyramidal or trigonal bipyramidal depending on the exact combination of ligands. The best models for the spectroscopic and chemical properties of the lipoxygenase iron site have all been sixcoordinate distorted octahedral complexes.39-41 It may be difficult to produce model complexes absent a protein scaffolding with the same level of distortion from ideal geometry as observed in the iron site of the cumene hydroperoxide treated crystals of lipoxygenase-3.

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Figure 11. Schematic representation of the iron sites in three crystallographically determined three-dimensional structures of lipoxygenase. Blue spheres, His N; orange sphere, Fe; red sphere, Asn O; pink sphere, OH or 13-HPOD O; structure, C-terminal Ile.

Because lipoxygenase-1 retains residual catalytic activity after treatment with cumene hydroperoxide, it is reasonable to suppose that the iron site in the treated crystals reflects the situation in the active enzyme and that the observed coordination geometry is a feature of the activated complex during catalysis. To illustrate the changes that could be taking place during catalysis, three schematic representations of the iron sites from the same perspective at various stages of the catalyzed reaction (native f peroxide oxidized f product complex) are presented in Figure 11. Significantly, treatment with cumene hydroperoxide resulted in an increase in the degree of iron atom exposure to the space where fatty acid binding takes place, as the bond angle His-523/Fe/OH expanded to almost 120°. This feature might have important consequences for the catalyzed reaction. Because of the mass of the proton, hydrogen tunneling can account for reactions taking place over only a limited distance (0.6-0.7 Å).42 The rearrangement that takes place in the lipoxygenase iron site upon peroxide-induced oxidation allows for close approach of the substrate to the cofactor. Other attributes of the iron site remain to be reconciled with the observed structure. For example, existing evidence implicates a relatively high redox potential for the cofactor (+0.5 to +0.7 V vs NHE) in order to be consistent with the properties of the catalyzed reaction. Iron complexes with nitrogen and carboxylate ligands such as iron(III)-EDTA and iron(III)-DTPA typically have much lower electrode potentials (ca. +0.1 V vs NHE).43 It is clear, however, from the accumulating crystallographic evidence regarding the lipoxygenase iron site that there is a lot of flexibility in the coordination geometry (Table 2). Protein dynamics may be a key component of the hydrogen tunneling mechanism.44 The three-dimensional structures indicate that fluctuations in the relative positions of the iron atom and its ligands can indeed take place. Experimental Section

(33) Ursby, T.; Adinolfi, B. S.; Al-Karadaghi, S.; De Vendittis, E.; Bocchini, V. J. Mol. Biol. 1999, 286, 189-205. (34) Nelson, M. J. J. Am. Chem. Soc. 1988, 110, 2985-2986. (35) Skrzypczak-Jankun, E.; Amzel, L. M.; Kroa, B. A.; Funk, M. O. Proteins Struct. Funct. Genet. 1997, 29, 15-31. (36) Wedekind, J. E.; Frey, P. A.; Rayment, I. Biochemistry 1995, 34, 1104911061. (37) Geeganage, S.; Frey, P. A. Biochemistry 1999, 38, 13398-13406. (38) Kitajima, N.; Tamura, N.; Amagai, H.; Fukui, H.; Moro-oka, Y.; Mizutani, Y.; Kitagawa, T.; Mathur, R.; Heerwegh, K.; Reed, C. A.; Randall, C. R.; Que, L.; Tatsumi, K. J. Am. Chem. Soc. 1994, 116, 9071-9085. (39) Ogo, S.; Wada, S.; Watanabe, Y.; Iwase, M.; Wada, A.; Harata, M.; Jitsukawa, K.; Masuda, H.; Einaga, H. Angew. Chem., Int. Ed. Engl. 1998, 37, 2102-2104. (40) Goldsmith, C. R.; Jonas, R. T.; Stack, T. D. P. J. Am. Chem. Soc. 2001, 124, 83-96. (41) Kim, J.; Zang, Y.; Costas, M.; Harrison, R. G.; Wilkinson, E. C.; Que, L. J. Biol. Inorg. Chem. 2001, 6, 275-284.

Lipoxygenases were extracted from soybeans cv. Resnik and purified by ammonium sulfate fractionation and chromatofocusing.45 Solutions of the enzymes in Tris HCl (0.1 M, pH 7.0) were stored at 4 °C. Samples of the proteins were transferred into the appropriate buffers using dialysis and/or diafiltration using 50 000 NMWCO membranes. Cumene hydroperoxide was purified from an old bottle of cumene by chromatography on silica. Cumene and cumene hydroperoxide were (42) Cha, Y.; Murray, C. J.; Klinman, J. P. Science 1989, 243, 1325-1330. (43) Engelmann, M. D.; Bobier, R. T.; Hiatt, T.; Cheng, I. F. BioMetals 2003, 16, 519-527. (44) Klinman, J. P. Pure Appl. Chem. 2003, 75, 601-608. (45) Funk, M. O.; Carroll, R. T.; Thompson, J. F.; Dunham, W. R. Plant Physiol. 1986, 82, 1139-1144. J. AM. CHEM. SOC.

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batch eluted with hexane and collected as fractions in 5% acetone in hexane (v/v), respectively. Stock solutions of the hydroperoxide were prepared by weight in absolute methanol and stored at -80 °C. The 13-HPOD was prepared from linoleic acid (Sigma) by the reaction catalyzed by lipoxygenase-1 at pH 9.2 and was purified by chromatography on silica.46 Samples were prepared in absolute methanol and stored at -80 °C. The concentration was determined by dilution in absolute methanol and UV-vis spectrophotometry using an  value of 23 000 L/mol-cm.47 Kinetic Measurements. The time course for the catalyzed reaction was obtained by following the formation of product, 13-HPOD, by the increase in absorbance at 234 nm at 25.0 °C in a temperature-controlled Cary 3E spectrophotometer. Linoleic acid solutions were prepared in sodium borate buffer (0.1 M, pH 9.0). The substrate (3.00 mL) and appropriate dilutions of cumene hydroperoxide in methanol (0.060 mL) were transferred to a cuvette and temperature equilibrated with magnetic stirring. An aliquot of the enzyme (0.010 mL, 0.5 µM) was added to initiate the reaction, and the absorbance at 234 nm versus time was saved as a csv data file. The maximum slope as an approximation of the steady-state rate was obtained using an interactive plotting program in Excel.48 Each determination was conducted three times consecutively, and the values were averaged. Each set of conditions was investigated at least three different times, and the values were averaged. The error represents the standard deviation found for a given set of conditions obtained on different occasions. Simulations of the maximum rate data were conducted with the interactive program DynaFit.49 The kinetic mechanism derived by Schilstra et al. was used.16 The rate/dissociation constants employed in the simulations were KP ) KP* ) 12 µM, KS ) KS* ) 12 µM, k1 ) 300 s-1, k2 ) 109 M-1 s-1, k3 ) 2300 s-1, and k4 ) 150 s-1. Preincubation of lipoxygenase-1 (1.00 mL, 0.5 µM) was carried out with varying concentrations of cumene hydroperoxide (0.02 mL in methanol) at 25 °C in sodium borate buffer. An aliquot (0.010 mL) of the preincubation solution was added to 3.00 mL of temperatureequilibrated (25.0 °C) substrate (linoleic acid, 100 µM, 2% methanol v/v) to initiate the reaction. The maximum slope was obtained for three consecutive determinations, and the values were averaged. The experiments were all replicated on separate occasions, and the values were averaged. Isothermal Titration Calorimetry. Titrations were conducted in a MicroCal VP-ITC microcalorimeter. The enzyme (0.008 mM) was in the cell in Tris HCl (0.1 M, pH 8.5). The concentration of the cell solution was determined by absorbance at 280 nm using an  value of 120 000 L/mol-cm.50 The hydroperoxides were in the titration syringe (0.100 mM) in the same buffer. The concentration of cumene hydroperoxide solutions was determined by absorbance at 257 nm using an  value of 190 L/mol-cm.51 The titrations consisted of 30 injections. A small heat of dilution contribution evident toward the end of each titration was subtracted from the data prior to fitting with Origin (one site). The experiments were carried out three times for both hydroperoxides. The reported error represents the standard deviation of three determinations on separate occasions. EPR Spectroscopy. The 9 GHz spectra were obtained on frozen solutions in a Bruker model ESP 300E spectrometer with an Oxford Instruments model ITC4 cryostat operating at 25 K. Microwave power was selected to avoid modulation broadening, typically 5 mW with 1 mT modulation amplitude. The samples were prepared by placing the appropriate quantity of cumene hydroperoxide solution in methanol (1, 2, 5, and 10 times the amount of lipoxygenase-1) in an Eppendorf (46) Funk, M. O.; Isaac, R.; Porter, N. A. Lipids 1976, 11, 113-117. (47) Graff, G.; Anderson, L. A.; Jaques, L. W. Anal. Biochem. 1990, 188, 3847. (48) Brault, P. A. M.S. Thesis, University of Toledo, 2000. (49) Kuzmic, P. Anal. Biochem. 1996, 237, 260-273. (50) Draheim, J. E.; Carroll, R. T.; McNemar, T. B.; Dunham, W. R.; Sands, R. H.; Funk, M. O. Arch. Biochem. Biophys. 1989, 269, 208-218. (51) Norrish, R. G.; Searby, M. H. Proc. R. Soc. London 1956, A237, 464-. 2014 J. AM. CHEM. SOC.

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tube. The methanol was removed using a stream of nitrogen. A sample of the enzyme (0.25 mL, 213.5 µM, 0.1 M Tris HCl pH 7.0) was added, and the contents were gently mixed. The samples were transferred to EPR tubes and frozen in liquid nitrogen. Mass Spectrometry. Mass spectra were obtained at the Ohio State University Mass Spectrometry and Proteomics Facility of the Campus Chemical Instrument Center on a Micromass Q-Tof II High Resolution electrospray ionization mass spectrometer. The instrument was configured to simultaneously collect MS and MS/MS data on the effluent from a Vydac 2.1 × 250 mm C18 Mass Spec HPLC column in a Hewlett-Packard Series 1100 HPLC. The chromatography column was eluted at 0.1 mL/min with a 120 min linear gradient from 1% to 65% 0.1% formic acid in acetonitrile starting with 0.1% formic acid in water. Lipoxygenase-1 (0.4 mL, 38.4 µM) was added to a solution of cumene hydroperoxide (2.27 µM, 9.6 mL) in 0.1 M Tris HCl pH 8.5, 2% methanol, (v/v). After 30 min at room temperature, the enzyme solution was transferred to 0.1 M ammonium bicarbonate by diafiltration using 50 ,000 NMWCO membranes. The final volume of each sample was 0.8 mL. The enzyme solutions were subjected to reduction, carboxamidomethylation, and trypsin digestion by closely following a published procedure.52 X-ray Crystallography. Lipoxygenase-3 solutions were dialyzed against Tris HCl buffer (0.1 M, pH 7.0), concentrated to 10 mg mL-1 and crystallized in a large batch technique by vapor diffusion in small concentric beakers cut to approximately 10 mm in height. The crystallization solution consisted of 600 µL of 20% PEG 8000 (w/v) in sodium citrate-phosphate buffer (0.05 M, pH 4.6), 100 µL of sodium phosphate buffer (0.1 M, pH 7.0), and 200 µL of deionized water. The solution was subsequently filtered through an ULTRAFREE-MC filter unit (0.45 µm) to remove any insoluble material. Microseeds were prepared by crushing available crystals in 20% PEG (w/v, 500 µL) using a pellet pestle mixer. The seed solution was diluted 1:100, and an aliquot (2-4 µL) was added to the crystallization solution. A small beaker (20 mm diameter × 10 mm height) containing the crystallization solution was placed in a larger beaker (40 mm diameter × 20 mm height) containing 5 mL of 20% PEG 8000 reservoir solution. The larger beaker was sealed with a glass plate and placed in an incubator at 23 °C. Crystals formed in 24-72 h and grew to be 0.5 mm long with 0.05 × 0.2 mm cross-section dimensions. Optically flawless crystals were transferred to a solution of cumene hydroperoxide prepared by combining 20 µL of a stock solution (0.375 M) in methanol with a solution of PEG 8000 (20%, w/v, 1 mL). Crystals were soaked for 12-24 h and subsequently equilibrated with 20% glycerol for cryoprotection. Glycerol was added incrementally over a period of 8 h, increasing the concentration gradually from 0.5% to 20%. A gradual increase in glycerol concentration prevented the lipoxygenase-3 crystals from going through a transition to a P lattice during flash-freezing as has been previously reported.27 Crystals were flash-cooled for data collection under a nitrogen cryostream at 98 K. Diffraction data were collected with a Quantum 210 ADSC at the IMCA-CAT beamline 17-ID of the Advanced Photon Source at Argonne National Laboratory. Crystals diffracted to a resolution limit of 1.95 Å with mosaicity values ranging from 0.9 to 1.4 Å. All reflections were reduced using the DENZO/SCALEPACK crystallographic data-reduction package.53 The unit cell parameters were a ) 111.001 Å, b ) 136.901 Å, c ) 61.512 Å, and β ) 96.236° with space group C2. The data set was 83% complete to 2.0 Å resolution with an overall Rmerge or 5.4%. The data collection statistics are provided in Table 3. The structure of cumene hydroperoxide treated lipoxygenase-3 was determined by molecular replacement using the room-temperature structure of the 13-HPOD-lipoxygenase-3 complex (PDB 1IK3) as (52) Stone, K. L.; Williams, K. R. A Practical Guide to Protein and Peptide Purification for Microsequencing, 2nd ed.; Academic Press: San Diego, 1993; Chapter 2. (53) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-326.

Enzyme Activation, Inactivation, and Inhibition Table 3. Data Collection and Space Group/Refinement Statistics for Cumene Hydroperoxide Treated Lipoxygenase-3 Crystals

beamline detector temperature (K) wavelength (Å) space group unit cell dimensions (Å), b (deg) no. of monomers in asymmetric unit Matthews coefficient, VM (Å3/Da) no. of images approximate exposure time (s) oscillation angle, Df (deg) crystal to detector distance (mm) resolution range (Å) no. of observations unique reflections completeness (%) mean I/s(I) Multiplicity Rsym (%) R-factor (50-2.0Å) Free R-factor (50-2.0Å) Average B-factor (Å2)

APS IMCA-CAT Beamline 17-ID Quantum 210 ADSC 98 1.00 C2 111.001, 136.901, 61.512, 96.236 1 2.4 (48.5%, v/v, solvent) 178 15 0.7 200 50-2.0 109 207 50 653 83.0 15.9 2.4 5.4 21.47 22.13 32.7

a The X-ray data were processed using the CCP4 package. Solvent content calculations were based on the expected solvent content of protein crystals.

starting coordinates for rigid body refinement with CNS.54 All water molecules, heteromolecules (13-HPOD), and the iron atom were omitted, resulting in an initial R factor of 38.0% (Rfree of 43.5%). Cryocooling resulted in an expected shrinkage of the unit cell dimensions. Simulated annealing followed by a cycle of positional and B-value refinement improved the R factor to 26.7% with Rfree of 28.4%.

ARTICLES The active site iron and the solvent structure were modeled and refined at this stage. Fo - Fc and 2Fo - Fc electron density maps were calculated showing excellent density for the main chain and good density for the side chains. The Ramachandran plot performed with PROCHECK indicated that 84.8% of residues fell within the sterically most favored region with an additional 12.1% and 2.1% within the allowed and generously allowed regions, respectively.55 Active site Fo - Fc and omit Fo - Fc maps were created, and a fifth ligand was modeled and refined. In addition, on the basis of mass spectrometry evidence, Fo - Fc and omit Fo - Fc maps were examined for evidence of side chain oxidation of Trp-519, Ser-564, Leu-565, and Val-566. Trp-519 showed very clear evidence for hydroxylation at the Cβ position at electron density levels of 3σ. A similar hydroxylation site was found at the Cβ position of Leu-565. The last stage of refinement included more B-value refinement and positional refinement with relaxation of bond length restraints for the hydroxylation sites. The B-values for the hydroxylation sites (occupancy ) 1.0) at Trp-519 and Leu-565 refined to 30.6 and 33.0, respectively, compared with an average B-value for the structure of 32.7. The B-value for the ironbound OH refined to 32.2 with a relaxed bond length of 2.05 Å. The final R factor for the resolution range, 50-2.0 Å, was 21.5% (Rfree 22.1%).

Acknowledgment. This research received financial support from the National Institutes of Health (GM 62140). We are grateful to Zhenwei Lu for carrying out the alignments of the three-dimensional structures. We thank the staff members of the Industrial Macromolecular Crystallography Association Collaborative Access Team (IMCA-CAT) for their assistance with data collection. JA0390855

(54) Brunger, A. T.; Adams, P. D.; Clore, G. M.; Delano, W. L.; Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Pannu, N.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr., Sect. D 1998, 54, 905-921.

(55) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. J. Appl. Crystallogr. 1993, 26, 283-291.

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VOL. 126, NO. 7, 2004 2015